Light entrains the suprachiasmatic nucleus (SCN), a circadian oscillator and a primary component of the mammalian circadian system. A subset of intrinsically photosensitive retinal ganglion cells (ipRGCs) express melanopsin, a novel vertebrate opsin, and pituitary adenylate cyclase-activating peptide (PACAP). These cells convey signals to the SCN providing information about ambient lighting conditions. However, the phototransduction system(s) used by these ipRGCs and the retinal neurons that are presynaptic to them are unknown. Moreover, the potential role of ipRGCs in regulating intra-retinal physiology is unexplored. The long term goals of this application are to provide an understanding of the phototransduction cascade used by ipRGCs, to describe the intra-retinal circuitry of this system of neurons to better understand their function, and to explore the role of melanopsin retinal ganglion cells in the regulation of intra-retinal dopamine. To study the phototransduction process, a highly enriched population of ipRGCs is generated using an immunopanning procedure. Isolated cells are examined using in vitro calcium imaging to observe light- stimulated calcium influx. The ipRGCs use melanopsin as a chromophore and neurons afferent to melanopsin-expressing cells will be identified using pseudorabies virus (PRV) as a transneuronal tracer. Mice in which Cre-recombinase is under the control of the melanopsin promoter will be infected with a conditional PRV that replicates only in neurons that express Cre-recombinase and in neurons in synaptic contact with the originally infected cells. Electron microscopy and calcium imaging will be used to explore the relationship between ipRGCs and the dopaminergic system of the retina. Finally, the role PACAP in modulating the effects of light on ipRGCs and the circadian system is examined using calcium imaging and in behavioral studies with PACAP null mice. Disturbances in the entrainment or phasing of our biological clock are responsible for abnormal phasing of sleep rhythms and may underlie serious affective disorders. Understanding the retinal neurons afferent to the SCN will aid in our ability to understand and treat these disturbances of phase. [unreadable] [unreadable] [unreadable]